Anti-cancer potential of substituted “amino-alkyl-rhodamine” derivatives against MCF-7 human breast cancer cell line

Breast cancer is the most prevalent diagnosed cancer among women and the main cause of morbidity and mortality. As for breast cancer, MCF-7 cells are an important candidate since they are widely utilized in research for estrogen receptor (ER)-positive breast cancer cell assays, and various sub-clones have been identified to reflect different classes of ER-positive tumors with varied levels of nuclear receptor expression. Rhodamines and its derivatives have shown a great interest over the past two decades due to their excellent structural and spectroscopic properties. Rhodamine derivatives have been widely investigated for their mitochondrial targeting and chemotherapeutic properties. Rhodamine derivatives, in particular, have been widely investigated for their therapeutic properties. In this regard, several studies have shown that rhodamine dye derivatives have promising in vitro and in vivo therapeutic efficacy. The present study deals with potential anticancer activity of few synthesized rhodamine derivatives against MCF-7 cell lines.


Introduction
Cancer is a broad term for a variety of diseases characterized by the uncontrollable division of abnormal cells with the ability to penetrate and dismantle normal body tissue. This generally accumulates as a mass or sheet of cells called as tumor. Tumors can be either malignant or noncancerous. A malignant tumor can develop and spread to other parts of the body, whereas a benign tumor can grow but not spread. Breast cancer can spread to other parts of the body via blood vessels, which is known as metastasis (Semina et al. 2022) . Based on the growth, it is being categorized as stages I, II, III, and IV. Although breast cancer spread to nearby lymph nodes, it can also spread through the body such as bone, lungs, liver, and brain and is called as metastasis or stage IV breast cancer and is the most advanced type of breast cancer (Rueda et al. 2019). Based on the spread pattern, breast cancer is of several forms, such as (i) ductal carcinoma (DC), (ii) ductal carcinoma in situ (DCIS), (iii) invasive or infiltrating ductal carcinoma (IDC), and (iv) invasive lobular carcinoma (ILC) (Garcia-Martinez et al. 2021).
Fluorescein and rhodamine dyes have been for many years the dyes of choice in fluorescence labeling (Hathaway et al. 2011). These xanthene-based dyes, which exhibit excellent spectroscopic characteristics such as large molar extinction coefficient, high emission quantum yield, absorption, and emission at longer wavelengths, are therefore tuned to facilitate in vitro and/or in vivo monitoring of various chemical and biological species exploiting their contrast structure-function relation. The fluorescence output of fluorescein type dyes is pH sensitive and bleaches quickly; thus, its applicability gets restricted in a variety of applications (Li et al. 2016). On contrary, rhodamine-based dyes become a preferable choice as labeling agents considering the tuneable fluorescence output, and ease of synthesis and functionalization. In progressive efforts to develop better photo-stable fluorescent labels, various parameters are to emerged to be taken in to consideration, such as capabilities of excitation and emission long-wave region, affinity for bio-conjugation where the absorption and emission maximums of bio-conjugate appears in the visible-near infrared (NIR) region of the spectrum, and stability in the operational pH condition where those are not affected by changes in pH between 4 and 10. These dyes find many uses in fluorescence microscopy, immunodiagnostics, in whole-body imaging, and microarray analysis (Battula et al. 2021). Cyanine-based dyes have a number of advantages over other dyes: the high molar extinction coefficient combined with a good quantum yield results in a very good brightness, which gives high sensitivity and direct labeling capability. Also, the addition of -C = C-units to the methine bridge between the two heterocyclic ring leads to a shift of ∼100 nm per unit in the excitation wavelength, leading to a wide range of multicolor dyes (Oza et al. 2016). The cyanines and related compounds, merocyanines, oxonols, and styryls have high photo-stability, bind by intercalating between the bases of nucleic acid molecules, and are not pH sensitive. These dyes have found use in DNA microarray technology to evaluate global changes in cell and tissue gene expression. Currently, small molecules like organic dyes, certain metal complexes, and their interaction with nucleic acids are of great interest in drug discovery (Toogood and Scrutton 2020). This present study involves screening of few rhodamine derivatives for their anti-cancer action in breast cancer cell line. To best of our knowledge, rhodamine B-based dyes have not extensively explored to evaluate the anti-cancer action in breast cancer cell line. In this context, rhodamine B 6-[3-(2-methoxy-5-methylphenylcarbamoyloxy)-9-azabicyclo[3.3.1] nonane-9-yl] hexylamide has been reported as the only known complex of amide derivative of rhodamine B to be used in the diagnosis of breast cancer (Wu et al. 2018). As the anti-carcinogenic medicines and their interactions with DNA play a key role in development of new cancer therapy treatment, it has been demonstrated that rhodamine derivatives binds in the grove of CT DNA. This binding affinity, interactions, and the structural aspects binding complex lead to pathway for natural and other synthetic dyes for implementation as therapeutic molecules (Hajian et al. 2012), rhodamine B derivatives in particular.

Materials and methods
Following the known protocols, all solvents were freshly distilled before to use for fluorescence measurements, and all reactions were conducted in a N 2 environment. Chromatographic separations were done by column chromatography using 100-200 mesh silica gel or neutral alumina.

Synthesis of 2
The precursor compound "amino-ethyl-rhodamine" was synthesized following a procedure reported earlier (Bag and Pal 2011a), which is also the precursor for synthesis of 1, 3, 4, 5, and 7, the previously reported compounds used in this investigation.

Synthesis of 5
To a stirring solution of freshly synthesized "amino-ethylrhodamine" (1.05 g, 2.06 mmol) in MeCN (40 mL), triethylamine (excess, 1.4 mL, 10.3 mmol) was added and stirred for 15 min, followed by addition of 2,6-bis-(bromomethyl) pyridine (0.55 g, 2.06 mmol) and the reaction mixture was heated to reflux for 24 h. The mixture, after bringing back to room temperature was filtered and the filtrate was evaporated to dryness under reduced pressure. Water (50 mL) was added to the residual mass and extracted with CHCl 3 (3 × 30 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and evaporated to dryness to obtain a reddish brown solid, which was isolated and purified by passing through a column (100-200 mesh silica gel) with chloroform as eluent.

Synthesis of 6
To a stirring solution of anthracene-9-carboxaldehyde (1.0 g, 5 mmol) in EtOH (20 mL), 1-(2-aminoethyl)-piperazine was added and allowed to react at room temperature for 24 h with constant stirring. The Schiff-base imine thus formed was reduced with NaBH 4 and was further refluxed for 2 h to ensure the complete reduction. The solvent was evaporated to dryness under reduced pressure. Water (50 mL) was added to the residual mass and extracted with CHCl 3 (3 × 30 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and evaporated to dryness to obtain a pale brown solid. It was further isolated and purified by passing through a column (100-200 mesh silica gel) with chloroform as eluent.

Anti-cancer studies
The standard protocol for anticancer activity testing uses Adriamycin (ADR) as a reference standard. The aim of this study was to evaluate our synthesized compounds (1-9), with the positive control ADR drug for their antiproliferative properties in vitro against MCF-7 cell lines of human cancer. The test substances' cytotoxic effects were assessed using MCF-7 human cancer cell lines. The activity of disintegrin-linker-melittin (DLM), which releases melittin after cleavage on the tumor cell surface, was taken for comparison in binding activity. Tables 1 and 2 summarize the LC50, TGI, and GI50 values obtained for each drug.
Test wells were compared to control wells on a plate by plate basis to determine percent increase. Percent growth was calculated as the ratio of the test well's average absorbance to the control wells' average absorbance multiplied by 100.
The cytotoxicity test findings are presented in three different formats. They are the lethal concentration (LC50), the growth inhibition factor (GI50), and overall growth inhibition (TGI). The LC50 value is the drug concentration that causes 50% cell death. The GI50 value is the drug concentration that inhibits cell growth by 50%. The TGI value is the drug concentration that causes complete cell growth inhibition.

Results and discussion
The rhodamine derivatives (1-5 and 7-9) which structurally differ through attached substituents at the spiro-cyclic amino-end were synthesized and characterized as per the standard protocol (Bag and Pal 2011a, b;Bag and Biswal 2012, Biswal and Bag 2013, 2015. The quaternary carbon peak at ~ 65 ppm in their 13 C NMR spectra inferred that these rhodamine derivatives (1-5 and 7-9) exist in their spirocyclic conformation. An aminoethylpiperazine appended anthracene derivative 6 was also synthesized and characterized, and taken in this study for comparison of activity with those of rhodamine derivatives. These compounds were observed to be stable over a 4-10 pH range (Bag and Pal 2011a, b;Bag and Biswal 2012), whereas their spiro-ring opening was observed under acidic environment (< 4.0 pH). The MCF-7 cells were treated with compounds at concentrations ranging from 10 to 80 µg/mL and the percentage control growth evaluated graphically.
The highest activity against MCF-7 cell line was that of compound 8, the bis-(aminoethyl)-piperazinyl appended rhodamine derivative, indicating − 42.1% control growth at 10 µg/mL concentration which is greater than any concentration of the positive control ADR (Table 1). Lower activities were noted for compound 3 with lowest percentage growth of 115.8% at 20 µg/mL concentration. Percentage control growth is graphically represented in Fig. 2. Compounds such as 1 and 9 showed unnoticeable growth control activity with low percentage of inhibition.
The GI50 value of compounds 2, 4, 5, 6, and 7 were < 10 µg/mL, which is comparable to that of positive control, ADR (Table 2). This indicates potent anticancer activity. Out of all these compounds, 6 exhibits < 10 µg/mL of both GI50 and TGI values, demonstrating highest anticancer potential.
Rhodamine bases and its derivatives have shown a great interest over the past two decades due to their excellent structural and spectroscopic properties. Battula et al. (2021) found that rhodamine bases have a dual impact, i.e., fluorescence and anticancer qualities, as well as greater cellular absorption by cancer cells compared to normal cells. Another study reported that anti-proliferative activity of rhodamine and its derivatives initiates cell death by blocking the G 0 /G 1 phase of the cell cycle, affecting the mitochondrial membrane potential, then entering the cells and specifically targeting lysosomes (Ma et al. 2018). These seem to contribute to the anticancer activity of the complexes.
It is imperative to understand that these compounds, being rhodamine B derivative in their closed form, have shown their anti-cancer properties. The ring-opened form of rhodamine derivatives are known to have mitochondrial targeting abilities, thus ascertained their chemotherapeutic properties. Few xanthene derivatives and rhodamine B derivatives are therefore known to be used as fluorescent dyes for cellular and mitochondrial membrane potential measurement (Yan et al. 2012). Moreover, rhodamine derivatives were shown to penetrate the mitochondrial membrane and to accumulate in this organelle, exhibiting high tumor cell selectivity (Johnson et al. 1980). Macașoi et al. (2020) investigated the mechanism of action of a rhodamine B-conjugated oleanolic acid derivative (RhodOA) which is a closed ring structure.
Consequently, the compound was tested on different human tumor cell lines (A375 melanoma, A549 lung adenocarcinoma, and MDA-MB-231 breast adenocarcinoma) and on a non-tumor cell line HaCaT human keratinocyte. The compound RhodOA reduced cell migration and produced condensation of cell nuclei and of actin fibers. Furthermore, impairment in melanoma cell mitochondrial function was observed, while the mitochondrial function of keratinocytes was left intact inducing mitochondrial mediated apoptosis in MCF-7, breast cancer cell line (Yan et al. 2012;Macașoi et al. 2020). These reports have established the mitochondrial targeting abilities of the rhodamine derivatives in their spiro-ring form, and a correlation therefore supports the anti-cancer activities of these rhodamine compounds here despite being in their ring-closed form. Nevertheless, our findings indicate that the highest activity against MCF-7 cell line was that of 8, indicating − 42.1% control growth at 10 µg/mL concentrations which is greater than any concentration of the positive control ADR. Previous literature data suggests that these dyes bind to the mitochondrial membrane and block oxidative phosphorylation, which serves as the major pathway of ATP production within the cell (Fearon et al. 1987). The lipophilic characteristic of these rhodamine dyes allows for enhanced penetration of dye into the cell through interactions with the phospholipid bilayer. The combination of both the lipophilic and cationic characteristics of these rhodamine dyes resulted in partially selective uptake of these dyes into the mitochondrion of cancer cells showing cytotoxic effects (Belostotsky et al. 2011;Modica-Napolitano and Apprile 2001). The higher cytotoxic activity of the compound 8 among all compounds under investigation against the breast cancer cell line therefore inferred to the physical and chemical nature of the compound, based on the results obtained and a correlation to the literature reports.

Conclusion
Results reported here demonstrate the synthesis and characterization of amine-based rhodamine could be promising candidates for cancer treatment. In vitro testing with the human breast cancer cell line MCF-7 treated with the desired chemicals revealed numerous alterations in cancer cell proliferation, according to the findings of this study. The highest significant anti-proliferating activity shown by rhodamine 8 can be linked with cell death, such as morphological alterations, and their numbers were reduced. The structure-function correlation of these compounds revealed that the rhodamine derivatives incorporating amino-piperazine substituents and nitro-aromatic substituents showed potential for anti-cancer activity. Therefore, molecular systems with structural similarity can further be designed, developed, and investigated for an optimal functional activity. Further, studies on these compounds and its significant effects on the expression of apoptosis genes such as P53, P21, and Mcl-1 can be an arena for future exploration (Rezaei-Seresht et al. 2019). As a result, we believe that these compounds are likely candidates for further investigation as therapeutic agents and adjuvant to established chemotherapeutic treatments in animal models of breast cancer. With subsequent cellular, molecular, and in vivo studies for diagnostic and bio-imaging purposes, a comprehensive mechanistic principle involving interactive activities should put further insight for the process.